Detection and Enumeration of Cytokine-Secreting Cells by FluoroSpot

The FluoroSpot assay is a development of the highly sensitive enzyme-linked immunospot (ELISpot) assay which enables functional measurement of immunity at the single-cell level. Both assays are performed in a 96-well format and measures the frequency of analyte-secreting cells, in ELISpot usually limited to one analyte per well due to the use of enzymes and precipitating substrates for detection. FluoroSpot, performed in a similar way as ELISpot, overcomes this limitation by detecting each analyte with an assigned fluorophore instead of an enzyme. By using readers equipped with fluorophore-specific filters, cells producing single or multiple cytokines can be identified simultaneously in the same well. This greatly facilitates the analysis of functionally distinct subpopulations in heterogenous cell samples, for example, the frequency of polyfunctional T cells, suggested to be of importance in various disease states. FluoroSpot maintains the simplicity and sensitivity of the ELISpot while taking the assay a step further towards a multiplex analysis and an in-depth understanding of the quality of an immune response. We describe here a 96-well plate method to analyze cells that have secreted up to four different cytokines simultaneously (Four-color Fluorospot).